The diversity of neurotransmitter receptors is far greater than had been anticipated by pharmacological and physiological studies. Two gene superfamilies encode all cloned neurotransmitter receptors: one is the G protein-coupled receptor superfamily, and the other is the ligand-gated ion channel superfamily (for reviews see Hall, 1987; Barnard, et al., 1987). All of the receptors that act through a G protein, for example, the muscarinic acetylcholine receptors, the dopamine receptors, and the .beta.-adrenergic receptors, are formed by a single polypeptide chain that is postulated to span the plasma membrane seven times. For each class of receptor, a gene family encodes closely related variants that have different pharmacological and physiological characteristics and different patterns of distribution in the nervous system.
In contrast to the G protein superfamily, ligand-gated ion channels are composed of more than one subunit. Expression studies have shown that the diversity of the ligand-gated receptors is due to the presence of the same subunits in different combinations. In addition, multiple genes encode each type of subunit. For example, three types of GABA.sub.A receptor subunits, .alpha., .beta., and .gamma., have been described, and at least for the .alpha. and .beta. subunits, several genes encode variant subtypes (Schofield, et al., 1987; Levitan, et al., 1988; Pritchett, et al., 1989). In transient transfection assays, functional receptors can be formed by either the .alpha. or the .beta. subunit alone or in pairwise combinations (Pritchett, et al., 1988). The presence of the .gamma. subunit, however, appears to be required for benzodiazepine sensitivity (Pritchett, et al., 1989).
One way to unravel the functional diversity of the nicotinic acetylcholine receptors (NACHRS) in the nervous system is to identify at the molecular level all of the different nAChR subunits. The pharmacology and single-channel characteristics of cloned subunits, expressed in various combinations in transfected cells or in Xenopus oocytes, can then be analyzed. By comparing these results with similar studies performed in vivo, a better understanding of the neuronal nicotinic pathways will become available. In pursuing this effort, the Molecular Neurobiology Laboratory at the Salk Institute for Biological Studies has isolated rat cDNA clones that identify four distinct neuronal nAChR subunits: .alpha.2, .alpha.3, .alpha.4 and .beta.2 (see U.S. Ser. Nos. 07/170,295, abandoned, 07/321,384, abandoned and Wada, et al., 1988; Boulter, et al., 1986; Goldman, et al., 1987; Deneris, et al., 1988). Recently, two additional clones that are closely related to the neuronal nAChRs but for which no function has yet been found have been identified. They are referred to as .alpha.5 an .beta.3 (U.S. Ser. Nos. 07/170,295, abandoned and 07/321,384, abandoned, and Boulter, et al., 1990; Deneris, et al., 1989). The present specification discloses another distinct neuronal nAChR subunit, beta4. In identifying the gene that encodes this subunit, rat genomic libraries were screened with neuronal nAChR cDNA probes. The genes for several of the previously described nAChR subunits were isolated, and their restriction maps were determined. A recombinant phage containing part of the gene for a novel nAChR subunit was also isolated. The primary structure of this subunit was deduced from the nucleotide sequence of a cDNA clone. Expression studies using Xenopus oocytes have shown that this subunit can combine with each of the neuronal .alpha.2, .alpha.3, and .alpha.4 subunits to form functional nAChRs.
The neuronal nAChR subunits are closely related and form a subgroup of the nAChR gene family that also includes the nicotinic receptors present in the Torpedo electric organ and at the vertebrate neuromuscular junction. The latter are formed by the pentameric assembly of four homologous subunits: two .alpha. ligand binding subunits and one each of the .beta., .gamma., and .delta. subunits (Reynolds and Karlin, 1978). There is evidence that during development, new nicotinic receptors containing an .epsilon. subunit instead of a .gamma. subunit are inserted at the neuromuscular endplate (Gu and Hall, 1988). Concomitant with this repopulation, an increase in channel conductance is observed (Sakmann and Brenner, 1978; Mishina, et al., 1986). Functional neuronal nAChRs can be produced by the coinjection into Xenopus oocytes of RNA encoding a .beta.2 subunit and one of either an .alpha.2, an .alpha.3 or an .alpha.4 subunit (U.S. Ser. Nos. 07/170,295, abandoned and 07/321,384, abandoned, and Boulter, et al., 1987; Wada, et al., 1988). Whiting and Lindstrom (1986, 1987) have proposed that the nicotinic receptors found in the peripheral and central nervous system are assembled from only two different subunits, an .alpha. and a .beta. subunit, in a yet undetermined stoichiometry. However, the possibility remains that neuronal nAChRs are composed of more than two distinct subunits.
As those skilled in the art will appreciate, an understanding of the molecular mechanisms involved in neurotransmission in the central nervous system is limited by the complexity of the system. The cells are small, have extensive processes, and often have thousands of synapses deriving from inputs from many different parts of the brain. In addition, the actual number of neurotransmitter receptors is low, making their purification difficult, even under the best of circumstances. Consequently, neither cellular nor biochemical approaches to studying neurotransmission in the central nervous system has been particularly fruitful. This is unfortunate because it is quite probable that the treatment of dementia, Alzheimer's disease and other forms of mental illness will involve modification of synaptic transmission with specific drugs.
The realization that the nicotinic acetylcholine receptors are much more diverse than previously expected offers an opportunity for a level of pharmaceutical intervention and a chance to design new drugs that affect specific receptor subunits. Such subtypes make it possible to observe the effect of a drug substance on a particular subtype. Information derived from these observations will allow the development of new drugs that are more specific, and therefore have fewer unwanted side effects.
In addition, the availability of these neuronal receptors makes it possible to perform initial in vitro screening of the drug substance. While it is true that the drug eventually has to work in the whole animal, it is probable that useful drugs are being missed because conventional screening is limited to average composite effects. Consequently, the ability to screen drug substances in vitro on a specific receptor subtype(s) is likely to be more informative than merely screening the drug substance in whole animals.
Both the receptor subunit DNA and the encoded protein(s) of the present invention can be used for drug design and screening. For example, the cDNA clone encoding the beta4 subunit, alone, or in combination with various alpha subunit clones or other subunit clones, now known or later to be discovered, can be transcribed in vitro to produce mRNA. This mRNA, either from a single subunit clone or from a combination of clones, can then be injected into oocytes where the mRNA will direct the synthesis of the receptor protein. Alternatively, the clones may be placed downstream from appropriate gene regulatory elements and inserted into the genome of eukaryotic cells. This will result in transfected cell lines expressing a specific receptor subtype, or specific combinations of subtypes. The derived cell lines can then be produced in quantity for reproducible quantitative analysis of the effects of drugs on receptor function.